Bacteria belonging to the genus Listeria are Gram-positive, non-spore forming, motile rods characterized in part by their capability for growth over a wide range of temperatures (4.degree. C. to 45.degree. C.). H. Zinsser, Microbiology (14th Edition) (Meredith Corp. 968) pp. 751-753. The taxonomic relationships between the genus and allied Gram-positive taxa are not clear. Furthermore, since all Listeria show a high degree of DNA relatedness and very close similarity in biochemical, phenotypic and protein characteristics, there is disagreement in the field as to relationships between the identified species within the genus. B. J. Wilkinson and D. Jones, J. Gen. Microbio. 98:399 (1977). J. McLauchlin, J. Appl. Bacterio. 63:1 (1987).
Improved characterization of the genus would have important medical consequences. Outbreaks of listeriosis in the United States have been reported from Listeria-contaminated food. For example, in 981, there was a major outbreak associated with cabbage, in 1983 with milk and in 1985 with contaminated cheese. Listeria infection in humans can cause a variety of symptoms ranging from cold-like to flu-like but is especially dangerous to a fetus where it induces a 50% mortality rate.
With the focus on disease prevention, rapid screening of food samples for Listeria is of foremost concern to the food industry. Under current regulations, the presence of viable cells of any Listeria species in foods is a cause for concern. Thus, there is a need, as a first step, to improve the capability to detect, identify and speciate all species of the genus.
With the need to improve the capability to speciate all species, there is also the need to better understand the pathogenicity, if any, with each particular species. Nearly all of the reported cases of human infections by bacteria belonging to the genus Listeria have been caused by Listeria monocytogenes. See McLauchlin, J. Appl. Bacterio. 63:1 (1987). However, instances have been reported in which Listeria ivanovii, Listeria innocua and Listeria seeligeri have caused disease in humans. It is thus, as a second step, important to determine whether specific strains are associated with disease.
The Food and Drug Administration (FDA) in the United States and comparable agencies in other countries have promulgated standard laboratory methods to detect the presence of Listeria in environment or food specimens (eg milk). These methdos involve culturing an appropriately prepared sample on microbiological media under conditions favorable for growth of these organisms and unfavorable for other bacteria. Detection of bacteria of the Listeria genus is attempted by examining the resulting colonies for morphological and biochemical characteristics, a process that typically is started 48 hours after acquisition of the sample and takes between 9-19 days to complete.
Newer methods of detecting Listeria include a) nucleic acid probes capable of binding to the nucleic acid of particular species, and b) antibodies capable of reacting with antigens specific to particular species. See e.g. A. R. Datta et al., Appl. Environ. Microbio. 54:2933 (1988) (probes to a fragment of a presumptive hemolysin gene of L. monocytogenes); J. Klinger, J. Assoc. Off. Anal. Chem 71:669 (1988) (nucleic acid hybridization assay for Listeria in foods); European Patent Application No. 0355147 (corresponding to U.S. Ser. No. 227,402 and 143,490) (a probe directed to the hemolysin gene in L. monocytogenes); European Patent Application No. 314294 (corresponding to U.S. Ser. No. 965,510) (probes to rRNA of L. monocyvtogenes); G. R. Siragusa and M. G. Johnson, Appl. Environ. Microbio. 56:1897 (1990) (monoclonal antibodies to an antigen shared with three Listeria species); J. A. Mattingly, J. Assoc. Off. Anal. Chem. 71:679 (1988) (antibody-based assay for detection of L. monocytogenes); European Patent Application No. 0303309 (corresponding to U.S. Ser. No. 36,619) (antibodies to heat-treated extracts of Listeria).
The present invention now provides a method capable of (1) detecting bacteria of the genus Listeria and (2) distinguishing between species of the genus Listeria. The method of the present invention has the advantage over previous methods in that it is easier, simpler and less expensive. It is also faster than the current biochemical characterization procedures which involve slow fermentation reactions detected with pH indicators.